How lipids and proteins cooperate to form functional membrane domains in cells remains a major unanswered question in cell biology. The lipid raft hypothesis has become a widely studied model for understanding this process. Rafts have been implicated in normal cellular functions ranging from membrane trafficking to cell signaling. They have also have been linked to a number of diseases and are thought to represent a major site for pathogen entry into cells. Current models propose that raft domains normally exist as nanoscale compositional fluctuations at steady state in cells, but can be stabilized to form functional rafts. However, how stabilized rafts assemble and function remains unclear. In this application, we propose to address these fundamental questions using the non-toxic membrane binding B-subunit of cholera toxin (CTxB) as a model. CTxB is a homopentamer that contains 5 binding sites for its glycolipid receptor, ganglioside GM1, itself a raft-associated molecule. Give its ability to cluster multiple GM1 molecules simultaneously, CTxB is widely regarded to serve as a raft crosslinker that builds stabilized raft domains. Moreover, the entry of cholera toxin int cells is thought to depend importantly on its targeting to raft-dependent, clathrin-independent endocytic pathways. However, the mechanisms by which these CTxB- stabilized domains form, how this process is regulated in cells, and how stabilized rafts are preferentially sorted into clathrin-independent endocytic carriers remain poorly understood. Here, we propose to investigate these questions though the following aims: 1) to test the hypothesis that CTxB must cluster raft-associated glycolipids to assemble stabilized raft domains; 2) to determine how toxin-stabilized rafts interact with the actin cytoskeleton; and 3) to determine if stabilized raft play an active role in targeting CTxB for uptake by clathrin independent endocytosis by mechanically deforming membranes. Successful completion of these aims will provide mechanistic insights into proteins and lipids cooperate to form stabilized rafts, how raft assembly is regulated by the cytoskeleton, and the role of stabilized rafts in endocytosis.